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Email: ytakada@ucdavis.edu Summary The integrins are a superfamily of cell adhesion receptors that bind to extracellular matrix ligands, cell-surface ligands, and soluble ligands.. On li

Yoshikazu Takada*, Xiaojing Ye* and Scott Simon †

Addresses: *Department of Dermatology, University of California Davis School of Medicine, Second Avenue, Sacramento, CA 95817, USA

†Department of Biomedical Engineering, College of Engineering, University of California, East Health Sciences Drive, Davis, CA 95616, USA Correspondence: Yoshikazu Takada Email: ytakada@ucdavis.edu

Summary

The integrins are a superfamily of cell adhesion receptors that bind to extracellular matrix ligands,

cell-surface ligands, and soluble ligands They are transmembrane αβ heterodimers and at least 18

α and eight β subunits are known in humans, generating 24 heterodimers Members of this family

have been found in mammals, chicken and zebrafish, as well as lower eukaryotes, including

sponges, the nematode Caenorhabditis elegans (two α and one β subunits, generating two integrins)

and the fruitfly Drosophila melanogaster (five α and one β, generating five integrins) The α and β

subunits have distinct domain structures, with extracellular domains from each subunit

contri-buting to the ligand-binding site of the heterodimer The sequence arginine-glycine-aspartic acid

(RGD) was identified as a general integrin-binding motif, but individual integrins are also specific

for particular protein ligands Immunologically important integrin ligands are the intercellular

adhesion molecules (ICAMs), immunoglobulin superfamily members present on inflamed

endo-thelium and antigen-presenting cells On ligand binding, integrins transduce signals into the cell

interior; they can also receive intracellular signals that regulate their ligand-binding affinity Here

we provide a brief overview that concentrates mostly on the organization, structure and function of

mammalian integrins, which have been more extensively studied than integrins in other organisms

Published: 1 June 2007

Genome Biology 2007, 8:215 (doi:10.1186/gb-2007-8-5-215)

The electronic version of this article is the complete one and can be

found online at http://genomebiology.com/2007/8/5/215

© 2007 BioMed Central Ltd

Gene organization and evolutionary history

The integrins are a superfamily of cell adhesion receptors that

recognize mainly extracellular matrix ligands and cell-surface

ligands, although some soluble ligands have been identified [1]

They are transmembrane αβ heterodimers, and at least 18 α

and eight β subunits are known in humans [2] (Figure 1; lists of

the integrin subunits present in mouse, chicken, zebrafish,

Caenorhabditis elegans, Xenopus laevis and Drosophila

melanogaster are given in Additional data file 1) Integrin α

and β subunits are totally distinct, with no detectable homology

between them; sequence identity among α subunits is about

30% and among β subunits 45%, indicating that both the α and

the β gene families evolved by gene duplication (Figure 2) In

humans, genes for both α and β subunits are located on various

chromosomes However, genes for integrins expressed in

leukocytes (subunits αL, αM, αD, and αX) are clustered at

16p11, while for those expressed in platelets and endothelial cells, the αIIb and β3 genes are at 17q21.32, and the α6, α4, and

αV cluster at 2q31 (Table 1) Some integrin 〈 subunits (〈1, 〈2,

〈10, 〈11, 〈M, 〈L, 〈D, and 〈X) contain a so-called I (insertion or interaction), or A, domain, while others do not The I-domain integrin α subunits are closely related to each other (Figure 2a) Also closely related to each other are the family of non-I-domain α subunits that recognize the RGD motif (αV, α8, α5, and αIIb) and the family of laminin-binding α subunits (α3, α6, and α7) Studies on integrin genes from lower and higher eukaryotes clearly indicate that integrin genes (both α and β) derived from a common ancestral gene by gene duplications A genomic analysis among 24 invertebrate and vertebrate species revealed that the α and β integrin structure, along with the inserted α I domain, has been highly conserved during the evolution of vertebrates [3]

Characteristic structural features

The crystal structures of human integrins αVβ3 [4,5] and

αIIbβ3 [6] show that the extracellular portion of an integrin

heterodimer consists of multiple domains (Figure 3a) The

headpiece of αVβ3, which contains the ligand-binding site,

consists of the β-propeller domain and the

plexin-sema-phorin-integrin (PSI) domain of the αV subunit, and the β

I-like, or βA, domain and the hybrid domain of the β

sub-unit The β-propeller domain contains seven repeats of

about 60 amino acids each that fold into a seven-bladed

β-propeller structure similar to the β subunit of a

hetero-trimeric G protein I domains contain a metal-ion-dependent

adhesive site (MIDAS) and I-like domains contain a

structurally similar metal-binding motif The RGD-binding

site is located at the interface between the β-propeller

domain and the β I-like domain and amino-acid residues

from the two domains interact directly with the RGD peptide

of a ligand [5] Mutagenesis studies have identified many

other amino-acid residues that are critical for ligand binding

[7,8] These residues are discontinuous in the primary

structure but are exposed on the surface of the headpiece

and generate the ligand-binding surface The crystal

structure of a complete integrin-ligand complex has not yet

been published, but by comparing the crystal structures of

RGD-bound and unbound forms it has been found that the

disulfide-linked loop structure in the β I-like domain

under-goes conformational changes (a movement of 1.5 Å towards

the RGD peptide), and the α helix 7 in the β I-like domain

moves downward on ligand binding [4,5] Also, the hybrid

domain swings outward upon integrin activation In the I-domain integrins, the I domain can be present in either an open (active) or a closed (inactive) conformation These are major conformational changes that affect ligand binding in the headpiece

Figure 2 Phylogenetic trees of integrin subunits Trees for (a) integrin α and (b)

integrinβ subunits are adapted from [58] and [59], respectively

Drosophila βPS

Human β7

Sea urchin βG

C elegans βPat3

Human β2

Human β1

Human β6

Human β8

Human β3

Human β5

Human β4

Sponge βPo1

Drosophila Fly βν

Coral βCn1

Crayfish β

(a)

(b)

αM αX αL αE α1 α2 α10 α11 Ascidian αHr1

I domain

α4 α9

αV α8 α5 αIIb

C elegans αF54F2.1

Drosophila αPS2 Sea urchin αSU2 Sea urchin αP

Ascidian αHR2 α6

α7 α3

Drosophila αPS1

C elegans αF54G8.3 Sponge α

Drosophila αPS3

Figure 1

The members of the human integrin superfamily and how they combine

to form heterodimeric integrins At least 18 α subunits and eight

β subunits have been identified in humans, which are able to generate

24 different integrins Integrin subunits that bind to each other to form a

heterodimer are connected by solid lines Each integrin has distinct

ligand-binding specificity and tissue and cell distribution

α4

α5

α6

α7

α8 α9 α10 α11

β4

αM αD

β7

β1

α2

β6

β8

The cytoplasmic tails of human integrin subunits are less

than 75 amino acids long (the β4 tail is an exception at a

length of approximately 1,000 amino acids, which includes

four fibronectin type III repeats) There is striking

homology among the β-subunit cytoplasmic tails, but the

α-subunit tails are highly divergent except for a conserved

GFFKR motif next to the transmembrane region, which is

important for association with the β tail A large number of

cytoskeletal and signaling proteins have been reported to

bind to β cytoplasmic tails and some have been found to

interact with specific α tails Most integrin β tails contain

one or two NPxY/F motifs (where x is any amino acid) that are part of a canonical recognition sequence for phosphotyrosine-binding (PTB) domains, which are protein modules present in a wide variety of signaling and cytoskeletal proteins Phosphorylation of the tyrosine (Y)

in the NPxY/F motif may represent a mode of regulating integrin interactions with other proteins at the cytoplasmic face of the plasma membrane The integrin tails recruit several proteins, such as talin, that bind actin filaments, and thus form a connection to the cytoskeleton, a connection that is essential for most, if not all,

Table 1

Human integrin subunits

Protein

ITGA2 α2 CD49b, α2 subunit of very late antigen 2 (VLA-2) NM_002203 5q23-q31 P17301

ITGA2B αIIb GTA, CD41, GP2B, HPA3, CD41b, GPIIb NM_000419 17q21.32 P08514

ITGA3 α3 CD49c, α3 subunit of VLA-3 NM_002204, NM_005501 17q21.33 P26006

ITGAE αE CD103, human mucosal lymphocyte antigen 1α NM_002208 17p13 P38570

ITGAL αL CD11a (p180), lymphocyte function-associated NM_002209 16p11.2 P20701

antigen 1 (LFA-1) α subunit

ITGAM αM Mac-1, CD11b, complement receptor 3 (CR3) subunit J03925, NM_000632, 16p11.2 P11215

ITGAV αV CD51, MSK8, vitronectin receptor α (VNRα) NM_002210 2q31-q32 P06756

ITGB1 β1 Fibronectin receptor β, CD29, MDF2, MSK12 BC020057 10p11.2 P05556

ITGB2 β2 Leukocyte cell adhesion molecule, CD18, CR3 subunit, NM_000211 21q22.3 P05107

CR4 subunit

ITGB3 β3 CD61; GP3A; GPIIIa, platelet glycoprotein IIIa NM_000212 17q21.32 P05106

integrin-mediated functions The structural basis for talin’s

unique ability to activate integrins through PTBs has been

defined [9] Structural data on integrins are mostly derived

from mouse and human and the structural basis for the

activation of integrins through their cytoplasmic domains

in other species is not yet known

Localization and function

Integrins function as traction receptors that can both

transmit and detect changes in mechanical force acting on

the extracellular matrix In mammals, some integrins are

limited to certain cell types or tissues: αIIbβ3 to platelets;

α6β4 to keratinocytes; αEβ7 to T cells, dendritic cells and

mast cells in mucosal tissues; α4β1 to leukocytes; α4β7 to a

subset of memory T cells; and the β2 integrins to

leukocytes Other integrins are widely distributed, such as

αVβ3, which is expressed on endothelium The RGD

sequence in fibronectin was originally identified as an

integrin-binding motif [10] and this and related sequences

in extracellular matrix molecules do act as integrin-binding motifs in vivo However, integrins also recognize many non-RGD sequences in their ligands, such as the tripeptide LDV in the immunoglobulin superfamily member vascular cell adhesion molecule 1 (VCAM-1), which is expressed on inflamed endothelium and is bound by α4β1 This pattern

of integrin recognition and activation appears to be conserved among most mammals studied

In regard to ligand specificity, the mammalian integrins can be broadly grouped into laminin-binding integrins (α1β1, α2β1, α3β1, α6β1, α7β1, and α6β4), collagen-binding integrins (α1β1, α2β1, α3β1, α10β1, and α11β1), leukocyte integrins (αLβ2, αMβ2, αXβ2, and αDβ2), and RGD-recognizing integrins (α5β1, αVβ1, αVβ3, αVβ5, αVβ6, αVβ8, and αIIbβ3) Individual integrins have unique ligand specificities (Table 2) They are further defined by those α subunits that can contain the I domain: these are α1, α2, α10, α11, αL, αM, αX, αD, and αE Non-I-domain subunits are α3, α4, α5, α6, α7, α8, α9, αV, and αIIb In I-domain integrins, the I I-domains play a central role in ligand binding and intercellular adhesion Integrin binding among invertebrate species is less well studied; RGD sequences have been found in species as diverse as sea urchins and amoebae, however, and integrins and their biochemical functions are likely to be highly conserved in metazoa, due to the essential nature of their function

Upon binding an extracellular ligand, integrins generate an intracellular signal and, conversely, their functioning can

be regulated by signals from within the cell [1] They serve

as transmembrane links between extracellular contacts (other cells or the extracellular matrix) and the actin microfilaments of the cytoskeleton, whose behavior integrins also regulate and modulate Many different proteins on the cytoplasmic side of the membrane, such as talin, vinculin, and ERM (ezrin, radixin, moesin) actin-binding proteins, act as linker proteins to connect the cytoplasmic domains of integrins to the cytoskeleton, resulting in complex interactions [1] Extracellular ligation

of integrins triggers a large variety of signal transduction events that modulate cell behaviors such as adhesion, proliferation, survival or apoptosis, shape, polarity, motility, haptotaxis, gene expression, and differentiation, mostly through effects on the cytoskeleton

The deletion of individual genes by gene knockout in mice shows that particular integrins play a critical role in development (the β1 integrins), vasculogenesis (αV inte-grins), lymphangiogenesis (α9β1), thrombus formation (αIIbβ3), the integrity of the skin (α6β4), and immune responses (the β2 integrins) (Table 3) Knockout of the gene for β3 enhanced tumorigenesis and angiogenesis [11,12], enhanced wound healing [13], and enhanced inflammation and atherosclerosis [14], suggesting that αVβ3 normally suppresses these processes

Figure 3

The extracellular region of a human integrin (a) The crystal structure

represents a net form of integrin αVβ3 with no bound RGD peptide

(Protein Data Bank (PDB) code 1JV2) [3,4] See PBD code 1L5G for the

RGD-bound form (b) The I (inserted or interactive) domain is present in

seven human α subunits between β-propeller repeats 2 and 3, and is

involved in ligand binding An I-like domain is present in all human integrin

β subunits along with four EGF-like repeats Both the I and I-like domains

have a Rossmann fold

l domain

l-domain-like structure

α

β

1 2 3 4 5 6 7

(b)

EGF-like repeats

Membrane

(a)

Inside-out signaling regulates integrin affinity

The affinity of individual integrins for their ligands in

mammals is tightly regulated by their heterodimeric

structure and by cytoplasmic signals from within the cell

(inside-out signaling) Integrins can be activated

intracellularly by signals from G-protein-coupled receptors

that lead to phosphorylation of the cytoplasmic domain of

the β subunit The association of the α and β cytoplasmic

tails appears to be required to maintain an integrin in the

inactive state; the association is disrupted by treatment

with agonists such as chemokines that are known to cause

integrin activation and which signal via G-protein-coupled

receptors [15] (Figure 4)

The cytoskeletal adaptor protein talin has been proposed to

play a role in regulating integrin affinity Binding of the

talin head region to the integrin β cytoplasmic tail causes

dissociation of the α and β tails and induces a

confor-mational change in the extracellular region that increases

its affinity for its ligand [15] Two models have been

proposed for this change in affinity In both, the inactive

integrin is in a bent conformation, with the headpiece

facing the membrane In the ‘deadbolt model’ the bent

conformation is maintained in an activated integrin, but

piston-like movements of the transmembrane regions

cause sliding of the extracellular stalks of the α and β

subunits, which disrupts the interaction between the

head-piece and the β stalk just beyond the membrane (the

deadbolt) [16] In the ‘switchblade model’, dissociation of

the α and β cytoplasmic and transmembrane regions leads

to dislocation of an epidermal growth factor (EGF)-like

repeat in the β stalk, which causes the head region to

extend outwards in a switchblade-like movement [17] In

both models, these proposed events correlate within

seconds with integrin ‘activation’, leading to

conformational changes in the ligand-binding pocket of the

headpiece that increase its affinity for ligand

The affinity directly regulates the nature of the ligand

binding and appears to tune the degree and kinetics of cell

adhesion In leukocytes, for instance, αLβ2 in an

intermediate-affinity state will interact with its ligand on

endothelium to help decelerate the leukocytes, which roll

slowly along the vessel wall but do not arrest (Figure 4a)

Conversion of αLβ2 to the high-affinity state by

intracellular signals from other receptors mediates their

complete arrest (Figure 4b) and signals cell polarization

and leukocyte movement across the post-capillary venule

wall into the inflamed tissue [18]

Outside-in signaling relays signals from the

extracellular environment

It has been proposed that on binding extracellular ligands,

mammalian integrins cluster in the membrane and

transduce signals to the interior of the cell (outside-in

signaling; Figure 4b) Extracellular ligand binding induces

conformational changes, including the outward swing of the hybrid domain, separation of the α and β ‘leg’ domains (Figure 3b), and separation of the transmembrane domains, that lead to the interaction of the cytoplasmic tails with intracellular signaling molecules [16] These include enzymes (for example, the focal adhesion kinase/c-Src, and the small GTPases Ras and Rho) and adaptors (for example, Cas/Crk and paxillin) that assemble within dynamic adhesion structures, including focal adhesions that bind cells to the extracellular matrix and podosomes (small foot-like extensions of plasma membrane) [15,19]

In this manner, the affinity of an integrin and its valence in binding ligands such as intercellular adhesion molecule-1 (ICAM-1) regulate the extent of outside-in signaling at the site of focal adhesive contacts (Figure 4) These contacts are active sites that transduce information such as the density of extracellular ligand or the magnitude and direction of extracellular forces on the cell Integrins can also be activated from the outside by the binding of divalent cations to the metal-ion-binding sites in the I and I-like domains in the α and β subunits, respectively

Binding of RGD-containing peptides or related compounds

to a site in the headpiece of the integrin heterodimers has been shown in crystal structures of αVβ3 [4,5] and αIIbβ3 [6] The binding site is composed of the β-propeller domain of the α subunit and the I-like domain of the β subunit The original crystal structure of integrin αVβ3 revealed a bent conformation of the head region associated with low affinity for ligand [4,5] It was therefore proposed that the bent form does not bind ligand or carry out outside-in signaling and that activated integrins have an extended form (see the switchblade model described above) Interestingly, it has been shown that the bent form

of αVβ3 can still bind to fibronectin [20] (see the deadbolt model described above) Several intermediate forms of integrin conformation have been postulated that confer ligand-binding affinities and a different activation and cell adhesion status from either the bent or the extended forms [21]

The medical potential of antagonists to human integrins

The interaction of integrins with their ligands is a major target for the development of therapeutic drugs A humanized anti-β3 antibody (abciximab) that blocks the binding of platelet integrin αIIbβ3 to fibrinogen has been used in the clinic to prevent thrombosis [22] A humanized anti-α4 antibody (natalizumab) that can block the α4β1-VCAM interaction or the α4β7-mucosal addressin cell adhesion molecule (MAdCAM) interaction on mucosal endothelium has been tested in clinical trials Natalizumab blocks leukocyte trafficking across the blood-brain barrier and thereby moderates inflammation in multiple sclerosis [23] Anti-α4 antibody is also effective in clinical trials in ameliorating inflammatory bowel diseases, for example, Crohn’s disease

Many RGD-based low-molecular-weight integrin

antagonists have been developed and some of them have

been approved as therapeutics (for example, eptifibatide and

tirofiban as inhibitors of αIIbβ3 to reduce platelet

aggregation and the formation of blood clots) [24] As more

becomes known about the relationship between integrin

three-dimensional structure and how this regulates affinity for

ligand and signaling into the cell, antagonists can be designed

that stabilize a specific conformation, thereby promoting or

blocking specific intercellular adhesion functions

Frontiers

Integrins are transmembrane molecules that are essential

for both embryonic development and immunological function

by binding to a wide variety of ligands, including extra-cellular matrix molecules and members of the immuno-globulin superfamily Their capacity to specifically recognize particular amino-acid motifs and regulate binding affinity to them lies in their heterodimeric structure This molecular design incorporates a remarkable ability to direct confor-mational changes initiated at the cytoplasmic domain, and also to signal extracellular ligand binding back to the inside

of the cell Much of our current knowledge of the myriad of functions attributed to ligand binding of a particular αβ pair comes from gene knockout studies in mouse or from rare hereditary disorders in humans Only a handful of crystal structures of integrins bound to their ligands have been solved From these data it appears that small variations in the particular structure or charge of a ligand (that is, down

Table 2

Ligand-binding specificities of human integrins

Integrins Ligands

α1β1 Laminin, collagen

α2β1 Laminin, collagen, thrombospondin, E-cadherin, tenascin

α3β1 Laminin, thrombospondin, uPAR

α4β1 Thrombospondin, MAdCAM-1, VCAM-1, fibronectin, osteopontin, ADAM, ICAM-4

α5β1 Fibronectin, osteopontin, fibrillin, thrombospondin, ADAM, COMP, L1

α6β1 Laminin, thrombospondin, ADAM, Cyr61

α8β1 Tenascin, fibronectin, osteopontin, vitronectin, LAP-TGF-β, nephronectin,

α9β1 Tenascin, VCAM-1, osteopontin, uPAR, plasmin, angiostatin, ADAM [25], VEGF-C, VEGF-D [26]

α10β1 Laminin, collagen

α11β1 Collagen

αVβ1 LAP-TGF-β, fibronectin, osteopontin, L1

αLβ2 ICAM, ICAM-4

αMβ2 ICAM, iC3b, factor X, fibrinogen, ICAM-4, heparin

αXβ2 ICAM, iC3b, fibrinogen, ICAM-4, heparin, collagen [27]

αDβ2 ICAM, VCAM-1, fibrinogen, fibronectin, vitronectin, Cyr61, plasminogen

αIIbβ3 Fibrinogen, thrombospondin, , fibronectin, vitronectin, vWF, Cyr61, ICAM-4, L1, CD40 ligand [28]

αVβ3 Fibrinogen, vitronectin, vWF, thrombospondin, fibrillin, tenascin, PECAM-1, fibronectin, osteopontin, BSP, MFG-E8, ADAM-15, COMP,

Cyr61, ICAM-4, MMP, FGF-2 [29], uPA [30], uPAR [31], L1, angiostatin [32], plasmin [33], cardiotoxin [34], LAP-TGF-β, Del-1

αVβ5 Osteopontin, BSP, vitronectin, CCN3 [35], LAP-TGF-β

αVβ6 LAP-TGF-β, fibronectin, osteopontin, ADAM

α4β7 MAdCAM-1, VCAM-1, fibronectin, osteopontin

αEβ7 E-cadherin

αVβ8 LAP-TGF-β

References are included for recently discovered ligands only Abbreviations: ADAM, a disintegrin and metalloprotease; BSP, bone sialic protein; CCN3,

an extracellular matrix protein; COMP, cartilage oligomeric matrix protein; Cyr61, cysteine-rich protein 61; L1, CD171; LAP-TGF-β, TGF-β latency-associated peptide; iC3b, inactivated complement component 3; PECAM-1, platelet and endothelial cell adhesion molecule 1; uPA, urokinase; uPAR, urokinase receptor; VEGF, vascular endothelial growth factor; vWF, von Willebrand Factor

to single atoms) can strongly influence the binding affinity

and the capacity of the integrin to maintain a conformation

that signals back into the cell This implies that ligand

binding can influence allosteric changes in the integrin,

which in turn dictate how the integrin reports on the

environment in which the cell finds itself Thus, integrins

serve as both sensors of their molecular surroundings and

effectors that conduct motile forces exerted by the cell’s

cytoskeleton and from the dynamic environment (that is,

shear forces within blood vessels) We are just beginning to

understand the structural and chemical basis of this

sensor-effector system A particularly exciting development is the

discovery of small molecules that bind tightly to the

ligand-binding pocket or to other domains and allosterically

stabilize integrin conformations that promote or antagonize

binding For instance, small molecules have been discovered

that can allosterically tune conformations of αLβ2 that favor

low, intermediate, and high-affinity binding In this manner,

it is possible to steer the adhesive response of a leukocyte in

a blood vessel to promote tethering and rolling, firm arrest,

or no binding at all It may be possible to apply such small molecules as therapeutics either to promote leukocyte recruitment at sites of infection or to block their accumu-lation in chronic inflammatory diseases such as in rheumatoid arthritis and psoriasis As more knowledge accumulates relating amino-acid sequence to common structural motifs associated with the allosteric control of ligand recognition and outside-in signaling to the cytoplasm,

it will become possible to design small molecules that target these critical domains

Additional data files

Additional data are available online with this article: Additional data file 1 contains tables of the integrin subunits present in the mouse, chicken, zebrafish, nematodes, Xenopus laevis, and D melanogaster

Table 3

Phenotypes of deletions of integrin subunits in the mouse

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Figure 4

Leukocyte recruitment to the endothelial surface (a) Binding of glycoprotein selectin ligands (yellow and purple) on the leukocyte to selectins (blue) on

the endothelial surface, and weak binding of low-affinity leukocyte integrins (green) to ICAMs (pale yellow) on the endothelium facilitates cell tethering and rolling This binding, together with signals from chemokines (pink), generates inside-out signals (yellow arrows) that shift the bound integrins to a

high-affinity ligand-binding state (b) Leukocyte arrest is mediated by clusters of high-affinity integrins (red) binding to ICAMs on the endothelial cells.

These focal clusters can themselves signal outside-in to affect functions such as cell polarization and migration

*

Chemokines Integin

Selectin ICAM

Chemokine receptor

Glycoprotein

selectin ligand

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